Lipidic particles have been shown to be efficient vehicles for many in vitro and in vivo applications. Lipidic particles complexed with DNA have been used in vitro (Felgner P. L., et al. Proc. Natl. Acad. Sci. USA 84, 7413-7417 (1987); Gao X. et al. Biochem. Biophys. Res. Commun. 179, 280-285 (1991)) and in vivo (Nabel E. G., et al. Science 249, 1285-1288 (1990); Wang C. et al. Proc. Natl. Acad. Sci. USA 84, 7851-7855 (1987); Zhu N., et al. Science 261, 209-211 (1993); Soriano P., et al. Proc. Natl. Acad. Sci. USA 80, 7128-7131 (1983)) for the expression of a given gene through the use of plasmid vectors. Formation of complexes of DNA with cationic lipidic particles has recently been the focus of research of many laboratories. In particular, lipofectin.TM. (Gibco BRL, Gaithersburg, Md.) has been successfully used for the transfection of various cell lines in vitro (Felgner P. L., et al. Proc. Natl. Acad. Sci. USA 84, 7413-7417 (1987)) and for systemic gene expression after intravenous delivery into adult mice (Zhu N., et al. Science 261, 209-211 (1993)).
Lipidic particles may be complexed with virtually any biological material. These particles may be complexed with proteins, therapeutic agents, chemotherapeutic agents, and nucleic acids and provide a useful delivery system for these agents. One such drug delivery system, gene therapy, is one such area which has produced promising results. In this area two different strategies have emerged: Gene therapy and oligonucleotide-based therapeutics. To be successful these two approaches must be mediated by an efficient "in vivo" transfer of the nucleic acid material to the target cells and there is a need to provide an efficient and safe delivery system of nucleic materials.
Gene therapy may involve the transfer of normal, functional genetic material into cells to correct an abnormality due to a defective or deficient gene product. Typically, the genetic material to be transferred should at least contain the gene to be transferred together with a promoter to control the expression of the new gene.
Viral agents have been demonstrated to be highly efficient vectors for the transfection of somatic cells. Retroviruses in particular have received a great deal of attention because they not only enter cells efficiently, but also provide a mechanism for stable integration into the host genome through the provirus. However, clinical use of retroviral vectors is hampered by safety issues. A first concern is the possibility of generating an infectious wild type virus following a recombination event. A second concern is the consequences of the random integration of the viral sequence into the genome of the target cell which may lead to tumorigenic event. In addition, as retroviruses would only complete their life cycle in dividing cells, a retroviral vector would be inefficient in targeting cells which are not dividing. DNA viruses such as adenoviruses are potential gene carriers but this strategy is limited in the size of the foreign DNA adenoviruses can carry and because of the restricted host range. However, the advantage of adenoviruses over retroviral vectors is their ability to infect post-mitotic cells.
Synthetic gene-transfer vectors have been subject to intense investigation since this strategy appears to be clinically safe. Potential methods of gene delivery that could be employed include DNA/protein complexes (Cristiano R. J., et al. Proc. Natl. Acad. Sci. USA 90, 2122-2126 (1993)) or lipidic particles (Nabel E. G., et al. Science 249, 1285-1288 (1990); Felgner P. L., et al. Proc. Natl. Acad. Sci. USA 84, 7413-7417 (1987); Wang C. et al. Proc. Nat. Acad. Sci. USA 84, 7851-7855 (1987); Gao X. et al. Biochem. Biophys. Res. Commun. 179, 280-285 (1991); Zhu N., et al. Science 261, 209-211 (1993); Soriano P., et al. Proc. Natl. Acad. Sci. USA 80, 7128-7131 (1983)). The genetic material to be delivered to target cells by these methods are plasmids. Plasmids are autonomous self-replicating extra chromosomal circular DNA. They can be modified to contain a promoter and the gene coding for the protein of interest. Such plasmids can be expressed in the nucleus of transfected cells in a transient manner. In rare events, the plasmids may be integrated or partly integrated in the cell host genome and might therefore be stably expressed. Episomal plasmid vectors are plasmids able to replicate in the nucleus of the transfected cells and may therefore be expressed in a total growing cell population. Plasmids have a promising potential considering the fact that they may be applied in combination with a synthetic vector as carrier and that gene therapy by this means may be safe, durable, and used as drug-like therapy.
Plasmid preparation is simple, quick, safe, and inexpensive representing important advantages over retroviral vector strategy. The successful use of this genetic tool for "in vivo" approaches to gene therapy will rely on the development of an efficient cell delivery system.
Retroviral vectors have been shown to be very efficient for gene therapy. However, their use for in vitro human gene therapy has several limitations. Retroviral vectors may, by insertional mutagenesis lead to activation of oncogenes and increase the frequency of malignant transformation. They will not transfect non dividing cells, and their stability and titer are adversely affected by large gene insert. Adenoviral vectors which give rise to transient expression are currently limited by a demonstrated toxicity in vivo. Presently, replication-compromised herpes simplex virus vectors have toxic effects on the cells they infect, thus limiting their use for human trials. These obstacles have led several laboratories to develop physical means of gene transfer such as the pneumatic DNA gun (Yang et al. 1990 Proc. Natl. Acad. Sci. USA 9568-72), direct DNA injection (Wolff et al. 1990 Science 247:1465-68), or liposome delivery vector (Fergner et al. Proc. Natl. Acad. Sci. USA 1987 84:7413-17).
The fact that viral vectors have limitations such as propensity for recombination, low titer, and induction of host immunity has initiated research into non-viral vectors. The delivery of plasmid DNA via synthetic carriers to cells "in vivo" by direct i.v. administration is appealing because of its simplicity and potential to reach a far greater number of cells than by an "ex vivo" approach. Although the efficiency of "in vivo" transfection of DNA plasmids is limited when compared to delivery by viral vectors, recent advances, especially in lipidic particle delivery, have demonstrated that non-viral gene transfer offers exciting potential, including its use in a clinical setting. More recent attempts to deliver gene or antisense oligonucleotides has provided a new impetus to lipid particle technology (Leonetti, J. P., et al. (1990) Proc. Natl. Acad. Sci. USA 87, 2448-2451; Burch, R. M. et al. (1991) J. Clin. Invest. 88, 1190-1196; Thierry, A. R. et al. (1992) Nucleic Acids Res. 20, 5691-5698; Smith, J. G., et al. (1993) Biochim. Biophys. Acta 1154, 327-340). One such approach is based on the formation of complexes of DNA with cationic lipidic particles. A few therapeutic clinical trial protocols using local administration of these complexes are ongoing but data on systemic administration is still poorly documented (Wang C. et al. (1987) Proc. Natl. Acad. Sci. USA 84, 7851-7855; Zhu, N. et al. (1993) Science 261, 209-211).
Several lipids have been used in attempts to prepare liposome-like particles. One such lipid mixture is Lipofectin.TM. which is formed with the cationic lipid DOTMA, N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium chloride, and DOPE, dioleylphosphatidyl ethanolamine at a 1:1 molar ratio. The lipidic particles prepared with this formulation spontaneously interact with DNA through the electrostatic interaction of the negative charges of the nucleic acids and the positive charges at the surface of the cationic lipidic particles. This DNA/liposome-like complex fuses with tissue culture cells and facilitates the delivery of functional DNA into the cells (Felgner P. L., et al. Proc. Natl. Acad. Sci. USA 84, 7413-7417 (1987)). New cationic lipid particles have been developed: Lipofectamine.TM. (Gibco BRL), composed of DOSPA, 2,3-dioleyloxy-N[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoracetate and DOPE at a 1:1 molar ratio. Lipofectace.TM. (Gibco BRL) composed of DDAB, dimethyidioctadecylammonium chloride and DOPE at a 1:1 molar ratio. DOTAP.TM. (Boehringer Mannheim, Ind.) is 1-2-dioleoyloxy-3 (trimethyl ammonia) propane.
Behr et al. (Proc. Natl. Acad. Sci. USA 86, 6982-6986 (1989); Barthel F., et al. DNA Cell Biol. 12, 6, 553-560 (1993)) have recently reported the use of a lipopolyamine (DOGS, Spermine-5-carboxy-glycinediotade-cylamide) to transfer DNA to cultured cells. Lipopolyamines are synthesized from a natural polyamine spermine chemically linked to a lipid. For example, DOGS is made from spermine and dioctadecylamidoglycine (Behr J. P., et al. Proc. Natl. Acad. Sci. USA 86, 6982-6986 (1989)). DOGS spontaneously condense DNA on a cationic lipid layer and result in the formation of nucleolipidic particles. This lipospermine-coated DNA shows high transfection efficiency (Barthel F., et al. DNA Cell Biol. 12, 6, 553-560 (1993)).
However, the above-described lipid compositions fail to produce liposomes. Rather, these investigations synthesized lipid particles, which are clusters of lipid molecules which have not formed at least a lipid bilayer membrane and therefore also lack an aqueous internal space. Since these particles are mere clusters of lipid molecules, the particles lack the ability to act as storage units for biologically active agents contained therein.
Therefore it is an object of the present invention to provide a liposomal composition capable of carrying internally biologically active agents.
It is an other object of the present invention to provide an efficient, stable and safe liposome-based delivery system for biologically active materials.
Yet another object of the present invention to provide a novel liposomal composition comprising a cationic lipopolyamine and a neutral lipid.
It is yet another object of the present invention to provide a method of transferring biologically active agents into cells and patients using the instant liposomal delivery system.
It is a further object of the present invention to provide a method of preparing liposomes, useful in providing efficient transfer therapy.
Yet a further object of the present invention relates to providing a method for long-term expression of a gene product from a non-integrated transgene in a patient.